Airway Remodelling in Children: When Does it Start?
Purpose of review: The review characterizes airway remodelling in childhood asthma and describes how early in life it is possible to detect, and possibly cure, the cellular and biochemical changes that characterize this event. This topic is timely and relevant since a variety of clinical and epidemiologic studies strongly suggest that in asthma, remodelling may start very early in life and that current prevention and treatment measures, including early avoidance measures and pharmaceutical interventions, are relatively ineffective in preventing the development of irreversible airway changes or in reverting them, once established.
Recent findings: Recent findings show that structural changes characterizing remodelling, such as subepithelial basement membrane thickening, epithelial cell disruption, protease/antiprotease imbalance and neoangiogenesis, are detectable in children with asthma and even in children with respiratory symptoms or with atopy, before a clear clinical diagnosis of bronchial asthma is made.
Summary: Identification of the early structural changes that may precede the development of asthma and of factors leading to permanent loss of lung function appear central to future asthma management.

In asthma, complex interactions between environmental exposure and genetic components generate chronic inflammatory processes within the airway and induce the development of airflow limitation and of airway hyperresponsiveness in response to a variety of stimuli.

The current concept of asthma pathogenesis in adult patients recognizes that independently from the aetiology, persistent inflammation is a key feature of this disorder, even in its mild form. Indeed, there is evidence that mediators released by inflammatory cells, recruited and activated within the airway tissue, have the potential to generate airway hyperresponsiveness and airflow limitation, i.e. the functional changes that are perceived by the patient as 'respiratory symptoms': cough, wheezing, breathlessness, and chest tightness. In predisposed individuals, persistent inflammation leads to 'remodelling'. This term refers to structural changes of the surface of the airway, induced by mediators released by the activated inflammatory cells, with shedding of epitheliocyte sheets in the bronchial lumen, and complex mechanisms, only partially understood, leading to tissue repair. These include activation of the surviving epithelial cells and of the underlying mesenchymal and vascular endothelial cells, with increased expression of surface adhesion molecules and release of proinflammatory cytokines, chemokines, and growth factors. As a consequence of these processes, two distinct results can be achieved: regeneration ad integrum, leaving no residual trace of the previous injury, or replacement of the 'normal' respiratory tract parenchyma by scar tissue, through the deposition of increased amounts of collagen (especially type III and V), fibronectin, and polysaccharides (such as hyaluronic acid).

Until recently, airway remodelling was considered to be a secondary phenomenon, developing late in the disease process as a consequence of persistent inflammation. It has therefore received less attention than the inflammatory component and its relevance to disease pathogenesis is still controversial. The importance of tissue remodelling as an early and consistent component of childhood asthma has been emphasized, however, in a recent biopsy study that described collagen deposition in the lamina reticularis and underlying fibroblast proliferation rather than eosinophil infiltration as consistent features of the disease.

There is evidence that the pathogenetic components of adult asthma may be shared by childhood asthma, at least in a proportion of patients. Indeed, postmortem and biopsy studies of children with asthma have shown epithelial cell disruption, eosinophilic infiltration, varying degrees of goblet-cell and submucous gland hyperplasia, subepithelial basement membrane collagen layer thickening, airway smooth muscle cell hypertrophy, and increase in the number and size of blood vessels in the airway wall. Airway inflammation and remodelling, however, have also been demonstrated in children with mild asthma and even in adolescents who outgrow their asthma, suggesting that inflammatory and repair processes may also be present in asymptomatic individuals.

Epidemiologic surveys have recently shown that a progressive decrease in lung function may also be seen in a significant proportion of young patients with asthma, strongly suggesting that remodelling may start very early in life, at least in predisposed individuals. In addition, longitudinal studies have demonstrated that current preventive measures and therapeutic interventions are relatively ineffective in preventing development of the irreversible airway changes or in reverting them, once established. These results have clearly indicated the need to evaluate in depth wheezing disorders in preschool children, to characterize the inflammatory events associated with the different asthma phenotypes in this age group. The aim is to better understand from an epidemiologic, clinical, immunologic and biochemical point of view the relative importance of the pathogenetic mechanisms involved in the induction of the acute and chronic injury to the airway structures, and in the modulation of the repair processes associated with the reversible versus the irreversible changes. Given the importance of early life events in the origins of asthma and remodelling, this 'temporal window' appears to be a key to gain new insights into mechanisms involved, leading to prevention and treatment interventions effective in altering the disease's natural history and severity.

Several studies in childhood asthma have addressed whether the various components of airway remodelling may be detected in infants and young children with 'preschool wheezing illness', a term currently used to include the different clinical phenotypes of asthma in this age group.

We briefly review here the most recent contributions specifically designed to evaluate subepithelial basement membrane thickening, epithelial cell disruption, protease/antiprotease imbalance, and neoangiogenesis, i.e. the increase in the number and size of blood vessels in the airway wall.

Among the different components of airway remodelling in asthma, increased depth of the reticular basement membrane (RBM) has been extensively investigated and qualitative observations have been confirmed in quantitative studies performed on bronchial biopsy specimens of children with mild to severe asthma. To demonstrate whether RBM thickening and the eosinophilic inflammation, characteristic of asthma in older children and adults, are present in symptomatic infants with reversible airflow obstruction, ultrathin sections of endobronchial biopsies were evaluated in three groups of infants: those with reversible airflow obstruction (group A); those with fixed airflow obstruction, without bronchodilator reversibility (group B); and those without airflow obstruction (group C). The median age of children belonging to the three groups was, respectively, 12 months (range 3.4-26 months) for group A, 12.4 months (range 5.1-25.9 months) for group B, and 11.5 months (range 3.4-24.3 months) for group C. The results showed that wheezing infants with reversible airflow obstruction had no evidence of RBM thickening or eosinophilia, since there were no significant differences in RBM thickness or inflammatory cell number between the three infant groups. Indeed, RBM thickness was lower in infants than in a group of older children (median age 10.3 years; range 6-16 years) with difficult asthma. Changes in RBM depth, however, may start very early, as shown by the study of Pohunek and coworkers. These authors evaluated children as young as 2 years of age with early respiratory symptoms, before a clear clinical diagnosis of bronchial asthma could be made. The same children were reevaluated 22-80 months after, to determine whether they had subsequently developed bronchial asthma. Examining bronchial biopsy specimens, more eosinophils in the bronchial mucosa and increased thickness of the subepithelial lamina reticularis were detected in children who had a diagnosis of bronchial asthma at follow-up. Thus changes characterizing airway remodelling may occur early in the natural history of the disease, even before diagnosis can be made on clinical symptoms. A common limitation in evaluating airway changes in childhood asthma is that invasive manoeuvres such as fiberoptic bronchoscopy with biopsy studies are problematic for a variety of reasons including ethical considerations.

A key question with obvious clinical implications is whether there are approaches, such as imaging techniques or biologic markers that can be obtained noninvasively, that are useful to gauge the presence of airway remodelling. De Blic and coworkers selected a group of children with severe asthma and a median age of 10.4 (9-13) years who underwent fiberoptic bronchoscopy with bronchial biopsy and bronchoalveolar lavage, measurement of exhaled alveolar nitric oxide levels, and high-resolution computed tomography (HRCT) scans. RBM thickness, number of intraepithelial inflammatory cells on bronchial biopsy, a variety of immune and inflammatory parameters on bronchoalveolar lavage, and alveolar nitric oxide concentrations were correlated with bronchial wall thickness on HRCT scans. They found a positive correlation between RBM thickening in bronchial biopsies and the bronchial wall thickness score, suggesting that HRCT scan might be considered as a valuable noninvasive technique for quantifying airway remodelling in children with severe asthma.

A rapidly growing field of research in respiratory medicine, particularly interesting for paediatrics, is the analysis of exhaled breath condensate (EBC). Although the condensate consists mostly of water vapour, it also contains aerosol particles released from the lung epithelial lining fluid, containing traces of nonvolatile solutes. The collection of EBC has been proposed as a simple, quick, safe, comfortable, and noninvasive technique suitable for assessment of biochemical and immunologic changes in the airways. With the background that cysteinyl leukotrienes (cysLTs) may stimulate human airway smooth muscle cell proliferation and, therefore, play an important role in airway remodelling, a study was designed to evaluate the relationship between cysLTs in EBC and RBM thickening in bronchial biopsies. Studying a group of children aged 4-15 years with moderate to severe persistent asthma, it was found that EBC cysLTs were significantly lower in those who were treated with the cysLT antagonist montelukast than in those who were not. Evaluating only the latter subgroup, however, a highly significant relationship between EBC cysLTs and RBM thickness was demonstrated, suggesting that biologic markers in EBC may be used to evaluate remodelling in asthma.

Epithelial damage and loss have been described as typical features of adult asthma, but their occurrence in childhood asthma has been supported only by indirect evidence, i.e. by the observation of an increased percentage of epithelial cells in bronchoalveolar lavage from children with asthma. To analyze the bronchial epithelium of normal children and children with asthma for the presence of markers of epithelial stress and repair, bronchial specimens obtained from children without asthma and from children with moderate or severe asthma, aged 5-15 years, were examined histochemically and immunohistochemically. The lamina reticularis of the biopsy sections obtained from children with asthma was found to be thicker than normal, with increased deposition of collagen III. In addition, an asthma-related increase in epidermal growth factor receptor (EGFR) expression by epithelial cells was found, a condition related in adult asthma to disease severity and indicating widespread epithelial stress or damage. Although there was no biochemical evidence of airway epithelial cell proliferation, a highly significant correlation was found between epithelial EGFR expression and the thickness of the lamina reticularis. Thus, in children with asthma, airway epithelium is stressed or injured and the phenotypical changes observed are associated with collagen deposition in the lamina reticularis. Since this epithelial damage was not associated with an adequate proliferative response, the hypothesis may be raised that the epithelium in children with asthma may be unable to repair itself after injury. Indeed, Barbato and coworkers found that, in addition to basement membrane thickness, epithelial loss was also increased in bronchial biopsies from children with mild or moderate asthma, aged 2-15 years, as compared with control subjects but also with atopic children without asthma. The observed epithelial damage was mainly due to loss of the columnar layer, while loss of both basal and columnar cells was present in few subjects and involved only a small percentage of the whole epithelium. In addition, stratifying the analysis according to age, it was observed that children with asthma who were younger than 6 years had increased epithelial loss and basement membrane thickening compared with control subjects of the same age. These observations together support the concept that 'cross-talking' between the damaged epithelial unit and the mesenchymal unit may play a significant role in the pathogenesis of remodelling in childhood asthma, triggering events that may promote increased collagen deposition, resulting in RBM thickening.

Proteolytic enzymes, such as the matrix metalloproteinases (MMPs), are continuously released in the airways and degrade extracellular matrix (ECM) components, including collagen type IV, a major constituent of the epithelial basement membrane. Turnover and remodelling of the ECM are highly regulated by the release of tissue inhibitor of MMPs (TIMP), since either uncontrolled proteolysis or excess collagen deposition may be associated with structural changes. Owing to their activities, MMPs may cleave cytokines and their receptors from the cell surface but also facilitate leukocyte migration between vascular endothelial cells and through the ECM components. In adult asthma, increased release of MMPs, such as MMP-9, by airway inflammatory and structural cells has been observed, possibly to balance the excessive deposition of ECM component that may be observed following injury. Evaluating children with asthma, it was found that the levels of MMP-9 and its inhibitor TIMP-1 were highly elevated in bronchoalveolar lavage cells, relative to the controls, about 30-fold and 35-fold, respectively. On the contrary, in another study performed in children with stable asthma undergoing elective surgical procedures, a significant reduction of the MMP-9 and of the ratio of MMP-9 to TIMP-1 was observed in bronchoalveolar lavage, as compared with controls. Thus, imbalance of MMPs and their inhibitors may occur in children with asthma, indicating protease/antiprotease activity disequilibrium and possibly reflecting early derangement of the metabolism of the ECM, which may play a significant role in airway remodelling.

Alterations of the vascular compartment, including increased vascularity associated with vasodilation and microvascular leakage, are also observed in adult asthma, and angiogenesis is acknowledged as an important component of the airway remodelling in this disorder. Remodelling of the airway vascular bed has been described in adult patients with asthma, with several quantitative studies reporting an increase in both total number of vessels and dilation of preexisting ones determining an enlargement of the total vascular area in the bronchial wall. New vessel formation is under the control of a variety of growth factors that may be synthesized and released as a result of the inflammatory events that characterize asthma. To test this hypothesis, the levels of two angiogenic factors, vascular endothelial growth factor (VEGF) and angiogenin, have been measured in sputum supernatants of children with asthma (mean age, 9.6 ± 3.5 years), during the acute attack and 6 weeks after start of therapy. As compared with controls, significantly higher sputum levels of VEGF and angiogenin were detected in children with asthma during the acute attack. VEGF and angiogenin levels were more elevated with increase in asthma severity, showing a significant negative correlation with FEV₁ values (forced expiratory volume in 1 second). Interestingly, after 6 weeks of inhaled corticosteroid therapy, a significant decrease in the two angiogenic factors levels was seen, without reaching the levels observed in normal controls. The hypothesis that in childhood asthma the injured epithelial surface of the airways may also trigger airway remodelling by releasing mitotic and fibrogenic growth factors, which may promote angiogenesis, is further supported by the study performed by Barbato and coworkers. These authors showed in bronchial biopsies from children with asthma, in addition to epithelial loss, basement membrane thickness, and eosinophil infiltration in the subepithelium, an increase in the numbers of vessels and in the percentage of area occupied by vessels in the subepithelium, in comparison with control children. The numbers of subepithelium vessels and eosinophils were also increased in atopic children without asthma, however, compared with controls, and were similar to those detected in patients with asthma. These observations suggest that inflammatory and structural changes occur early in the natural history of asthma, but they also emphasize that some of these pathologic lesions may be associated with atopy even in the absence of asthmatic symptoms. Whether these structural changes precede the development of asthma in atopic children who do not have asthma is an interesting hypothesis that deserves further investigation.